title: Isolation and genetic characterization of human coronavirus NL63 in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells.
abstract: BACKGROUND
  Cryopreserved primary human renal proximal tubule epithelial cells (RPTEC) were obtained from a commercial supplier for studies of Simian virus 40 (SV40). Within twelve hrs after cell cultures were initiated, cytoplasmic vacuoles appeared in many of the RPTEC. The RPTEC henceforth deteriorated rapidly. Since SV40 induces the formation of cytoplasmic vacuoles, this batch of RPTEC was rejected for the SV40 study. Nevertheless, we sought the likely cause(s) of the deterioration of the RPTEC as part of our technology development efforts.
  
  
  METHODS
  Adventitious viruses in the RPTEC were isolated and/or detected and identified by isolation in various indicator cell lines, observation of cytopathology, an immunoflurorescence assay, electron microscopy, PCR, and sequencing.
  
  
  RESULTS
  Cytomegalovirus (CMV) was detected in some RPTEC by cytology, an immunofluorescence assay, and PCR. Human Herpesvirus 6B was detected by PCR of DNA extracted from the RPTEC, but was not isolated. Human coronavirus NL63 was isolated and identified by RT-PCR and sequencing, and its replication in a fresh batch of RPTEC and another type of primary human kidney cells was confirmed.
  
  
  CONCLUSIONS
  At least 3 different adventitious viruses were present in the batch of contaminated RPTEC. Whereas we are unable to determine whether the original RPTEC were pre-infected prior to their separation from other kidney cells, or had gotten contaminated with HCoV-NL63 from an ill laboratory worker during their preparation for commercial sale, our findings are a reminder that human-derived biologicals should always be considered as potential sources of infectious agents. Importantly, HCoV-NL63 replicates to high titers in some primary human kidney cells.
authors.affiliation: Environmental and Global Health, College of Public Health and Health Professions, University of Florida, Box 100188, Gainesville, FL 32610-0188, USA. jlednicky@phhp.ufl.edu
authors.forename: John A
authors.initials: JA
authors.lastname: Lednicky
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Waltzek
authors.forename: Elizabeth
authors.initials: E
authors.lastname: McGeehan
authors.forename: Julia C
authors.initials: JC
authors.lastname: Loeb
authors.forename: Sara B
authors.initials: SB
authors.lastname: Hamilton
authors.forename: Maya C
authors.initials: MC
authors.lastname: Luetke
mesh_terms: D002460:Cell Line; D058957:Coronavirus NL63, Human / Q000302:isolation & purification* / Q000502:physiology*; D003584:Cytological Techniques; D003587:Cytomegalovirus / Q000302:isolation & purification; D004847:Epithelial Cells / Q000821:virology*; D005455:Fluorescent Antibody Technique; D015654:Herpesvirus 6, Human / Q000302:isolation & purification; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics; D017422:Sequence Analysis, DNA; D014779:Virus Replication
chemical_list: D012367:RNA, Viral